4. TRD#3: An automated and streamlined pipeline for in-situ molecular microscopy Abstract Our goal for this TRD is to integrate all of the techniques required for in situ molecular microscopy into a robust and reliable pipeline, and make it available to the scientific community. In situ molecular microscopy, that is reconstructing molecules inside cells or tissue, requires the integration of several challenging methods and techniques. Regions of interest need to be identified using cryo light micrsocopy (cryoLM), the specimen needs to be thinned down around this region of interest using a focused ion beam scanning electron microscope (FIB-SEM) in order to make it amenable to subsequent cryo electron tomography (cryoET). We will develop a variety of approaches for specimen preparation including some that are high-risk but of potentially high-impact. We will expand the Appion pipeline to integrate and track images, analysis and metadata across all three imaging modalities. Providing streamlined and transparent access to this pipeline is an essential aspect of making these methods accessible to general users. We will incorporate the ability to integrate light microscopy and SEM images into our image tracking database and enable relevant image processing techniques using our web based pipeline; the database that underlies both Leginon and Appion will be key to tracking these multiple imaging modalities and related actions and metadata. Several important biological projects will drive improvements in the design, development, automation and streamlining of this pipeline, and we anticipate a range of Collaborative and Service projects will make excellent use of the pipeline as soon as it is available. We will work closely with several groups with deep experience with in situ microscopy (Elizabeth Villa, Carolyn Larabell, and Michael Marko) to guide this project in the right direction. We will hold an annual weeklong workshop to train users in these methods and we will help to disseminate the overall pipeline to other labs in the scientific community, just as we do now for our Leginon and Appion systems.